A simple method for DNA sequencing devised by Frederick Sanger where a collection of DNA fragments are synthesized by means of controlled interruption of enzymatic replication. Four DNA synthesis reactions are carried out simultaneously with the strand whose sequence is to be determined being used as the template. The reaction mixture consists of regular deoxynucleotides and DNA Polymerase along with a small amount of one labelled dideoxy nucleotide analog being added to each of the four reaction mixtures. A primer is added to begin the DNA synthesis and strand elongation continues until a dideoxy analog gets added instead of the regular dNTP. Chain termination occurs at this stage due to the absence of a 3’ OH group for formation of the next phosphodiester bond. The synthesized strands are separated from each other, after which the differentially labelled strands of various lengths are separated by electrophoresis. The smallest fragments move further in the gel while the larger fragments remain close to the point of application. The different fluorescent labels of each ddNTP can then be detected by scanning the gel with a beam of laser. The output sequence obtained is complementary to the template strand, which can be used to deduce the original desired template sequence.

Genomic DNA is cleaved using a suitable restriction endonuclease and the fragments inserted into bacterial artificial chromosome vectors. These vectors enable the DNA fragments to be amplified. The genomic DNA fragments of the library are then organized into a physical map, after which individual clones are selected for sequencing. The selected BAC is amplified and these clones are sequenced using the Sanger’s chain termination method. The sequence of the clone is then deduced by aligning them based on their overlapping regions. The entire genomic sequence is then obtained once each BAC is sequenced in this manner.

In Pyrosequencing multiple round of nucleotide addition are carried out on the immobilized template DNA using DNA Polymerase in the presence of ATP sulfurylase, luciferase and the nucleotide degrading enzyme apyrase. The release of an equal amount of pyrophosphate is determined by its conversion to ATP by ATP sulfurylase which is determined by the release of light on reaction with luciferase. The amount of light produced is determined by means of a CCD camera, which is used to determine the addition of nucleotides & therefore the sequence of the template DNA.

The nanopore sequencing offers a label-free approach for DNA sequencing. An exonuclease cleaves the single stranded DNA, one base at a time to release the nucleoside monophosphates. These NMPs pass through the nanopore under an applied potential, which is covalently coupled to an adapter molecule. Continuous movement of NMPs through the nanopore results in characteristic fluctuation of electric current that enables detection of various nucleotide bases.

Illustration: Genome sequencing projects

Genome sequencing projects have aimed to elucidate the complete genome sequence of organisms. The DNA sequences are identified by the shotgun sequencing technique and then aligned using suitable software to provide the complete genome sequence. The genome sequence of a large number of prokaryotic and eukaryotic organisms has been successfully deduced. The immense amount of information provided by the human genome motivated researchers to understand the nature and content of genetic material in great detail. The shotgun approach was the fundamental technique used for large scale sequencing of the human genome, which also makes use of Sanger’s sequencing. Progress made in sequencing was very rapid and by 2001, a draft of the sequence was ready covering around 83% of the genome. The genome sequencing studies successfully provided many novel findings about the human genome.